The present invention relates generally to a newly identified protein tyrosine phosphatase and related products and methods.
The following description of the background of the invention is provided to aid in understanding the invention, but is not admitted to describe or constitute prior art to the invention.
Cellular signal transduction is a fundamental mechanism whereby extracellular stimuli are relayed to the interior of cells and subsequently regulate diverse cellular processes. One of the key biochemical mechanisms of signal transduction involves the reversible phosphorylation of proteins. Phosphorylation of polypeptides regulates the activity of mature proteins by altering their structure and function. Phosphate most often resides on the hydroxyl moiety (xe2x80x94OH) of serine, threonine, or tyrosine amino acids in proteins.
Enzymes that mediate phosphorylation of cellular effectors generally fall into two classes. The first class consists of protein kinases which transfer a phosphate moiety from adenosine triphosphate to protein substrates. The second class consists of protein phosphatases which hydrolyze phosphate moieties from phosphoryl protein substrates. The converse functions of protein kinases and protein phosphatases balance and regulate the flow of signals in signal transduction processes.
Protein kinases and protein phosphatases are generally divided into two groupsxe2x80x94receptor and non-receptor type proteins. Most receptor-type protein tyrosine phosphatases contain two conserved catalytic domains, each of which encompasses a segment of 240 amino acid residues. Saito et al., 1991, Cell Growth and Diff. 2:59-65. Receptor protein tyrosine phosphatases can be subclassified further based upon the amino acid sequence diversity of their extracellular domains. Saito et al., supra; Krueger et al., 1992, Proc. Natl. Acad. Sci. USA 89:7417-7421.
Protein kinases and protein phosphatases are also typically divided into three classes based upon the amino acids they act upon. Some catalyze the addition or hydrolysis of phosphate on serine or threonine only, some catalyze the addition or hydrolysis of phosphate on tyrosine only, and some catalyze the addition or hydrolysis of phosphate on serine, threonine, and tyrosine.
Tyrosine phosphatases can down-regulate the catalytic activity of protein kinases involved in cell proliferation and are therefore thought to be possible anti-cancer proteins. Protein phosphatases with inappropriate activity are also involved in some types of cancer. Because abnormally elevated levels of cell proliferation are associated with receptor and non-receptor protein kinases with unregulated activity, protein phosphatase-catalyzed dephosphorylation of a protein kinase can down-regulate kinase activity and thereby decrease the rate of cell proliferation.
In addition to their role in cellular proliferation, protein phosphatases are thought to be involved in cellular differentiation processes. Cell differentiation occurs in some cells upon nerve growth factor (NGF) or epidermal growth factor (EGF) stimulation. Cellular differentiation is characterized by rapid membrane ruffling, cell flattening, and increases in cell adhesion. Chao, 1992, Cell 68:995-997.
Alignment of primary amino acid sequences of known PTPs shows that their catalytic domains share common amino acid sequences. This observation has facilitated efforts of cloning protein phosphatases from multiple organisms and tissues. Probing cDNA libraries with polynucleotides complementary to cDNA encoding protein phosphatase consensus sequences has identified cDNAs resembling protein phosphatase sequences via the polymerase chain reaction (PCR). Some polypeptide molecules encoded by these cDNAs have tyrosine phosphatase activity.
The present invention relates to nucleic acid molecules encoding a newly identified protein tyrosine phosphatase named PTP20, nucleic acid molecules encoding portions of the fill length protein, nucleic acid vectors harboring such nucleic acid molecules, cells containing such nucleic acid vectors, purified polypeptides encoded by such nucleic acid molecules, antibodies to such proteins and polypeptides, and methods of identifying compounds that bind PTP20 or abrogate its interactions with natural binding partners. Also disclosed are methods for diagnosing abnormal conditions in an organism with PTP20 related molecules or compounds. The nucleic acid molecules, nucleic acid vectors, host cells, polypeptides, and antibodies may be produced using the information provided herein in conjunction with well known and standard techniques used currently in the art.
The present invention is based in part upon the isolation and characterization of nucleic acid molecules encoding a novel protein phosphatase designated PTP20. PTP20 regulates growth factor stimulation of cellular differentiation. PTP20 is thought to be involved in cellular differentiation, as its over-expression in rat pheochromocytoma cells (PC12) is associated with increased rates of differentiation. Various treatments of neural cancers as well as neural damage are thus provided based on the discovery of PTP20 and its role in these disorders.
Thus in a first aspect, the invention features an isolated, enriched, or purified nucleic acid molecule encoding a PTP20 polypeptide.
The term xe2x80x9cisolatedxe2x80x9d, in reference to nucleic acid molecules, indicates that a naturally occurring sequence has been removed from its normal cellular environment. Thus, the sequence may be in a cell-free solution or placed in a different cellular environment. The term does not imply that the sequence is the only nucleotide chain present, but that it is essentially free (about 90-95% pure at least) of non-nucleotide material such as chromosomal DNA or proteins.
The term xe2x80x9cenrichedxe2x80x9d, in reference to nucleic acid molecules, means that the specific DNA or RNA sequence constitutes a significantly higher fraction (2-5 fold) of the total DNA or RNA present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. A person skilled in the art could enrich a nucleic acid mixture by preferentially reducing the amount of other DNA or RNA present, or preferentially increasing the amount of the specific DNA or RNA, or both. However, nucleic acid molecule enrichment does not imply that there is no other DNA or RNA present, the term only indicates that the relative amount of the sequence of interest has been significantly increased. The term xe2x80x9csignificantlyxe2x80x9d qualifies xe2x80x9cincreasedxe2x80x9d to indicate that the level of increase is useful to the person performing the recombinant DNA technique, and generally means an increase relative to other nucleic acids of at least 2 fold, or more preferably at least 5 to 10 fold or more. The term also does not imply that there is no DNA or RNA from other sources. Other DNA may, for example, comprise DNA from a yeast or bacterial genome, or a cloning vector. In addition, levels of mRNA may be naturally increased relative to other species of mRNA when working with viral infection or tumor growth techniques. The term xe2x80x9cenrichedxe2x80x9d is meant to cover only those situations in which a person has intervened to elevate the proportion of the desired nucleic acid.
Most methods of recombinant nucleic acid manipulation require that these molecules are in a purified form. The term xe2x80x9cpurifiedxe2x80x9d, in reference to nucleic acid molecules does not require absolute purity (such as a homogeneous preparation); instead, it represents an indication that the sequence is relatively more pure than in its cellular environment (compared to the natural level this level should be at least 2-5 fold greater, e.g., in terms of mg/ml). The claimed DNA molecules obtained from clones could be obtained directly from total DNA or from total RNA. cDNA clones are not naturally occurring, but rather are preferably obtained via manipulation of a partially purified, naturally occurring substance (messenger RNA). The construction of a cDNA library from mRNA involves the creation of a synthetic substance (cDNA). Individual cDNA clones can be isolated from the synthetic library by clonal selection of the cells carrying the cDNA library. Thus, the process which includes the construction of a cDNA library from mRNA and isolation of distinct cDNA clones yields an approximately 106-fold purification of the native message. Thus, purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is favored in these techniques.
The term xe2x80x9cnucleic acid moleculexe2x80x9d describes a polymer of deoxyribonucleotides (DNA) or ribonucleotides (RNA). The nucleic acid molecule may be isolated from a natural source by cDNA cloning or subtractive hybridization or synthesized manually. The nucleic acid molecule may be synthesized manually by the triester synthetic method or by using an automated DNA synthesizer.
The term xe2x80x9ccDNA cloningxe2x80x9d refers to hybridizing a small nucleic acid molecule, a probe, to genomic cDNA that is bound to a membrane. The probe hybridizes (inds) to complementary sequences of cDNA.
The term xe2x80x9ccomplementaryxe2x80x9d describes two nucleotides that can form multiple favorable interactions with one another. For example, adenine is complementary to thymidine as they can form two hydrogen bonds. Similarly, guanine and cytosine are complementary since they can form three hydrogen bonds. Thus a xe2x80x9ccomplementxe2x80x9d of a nucleic acid molecule is a molecule containing adenine instead of thymine, thymine instead of adenine, cytosine instead guanine, and guanine instead of cytosine. Because the complement contains a nucleic acid sequence that forms optimal interactions with the parent nucleic acid molecule, such a complement binds with high affinity to its parent molecule.
The term xe2x80x9chybridizexe2x80x9d refers to a method of interacting a nucleic acid molecule (e.g., a nucleic acid probe) with a DNA or RNA molecule in solution or on a solid support, such as cellulose or nitrocellulose. If a nucleic acid probe binds to the DNA or RNA molecule with high affinity, it is said to xe2x80x9chybridizexe2x80x9d to the DNA or RNA molecule. As mentioned above, the strength of the interaction between the probe and its target can be assessed by varying the stringency of the hybridization conditions. Various low or high stringency hybridization conditions may be used depending upon the specificity and selectivity desired. Stringency is controlled by varying salt or denaturant concentrations. Those skilled in the art will recognize how such conditions can be varied to vary specificity and selectivity. Under highly stringent hybridization conditions only highly complementary nucleic acid sequences hybridize. Preferably, such conditions prevent hybridization of nucleic acids having one or two mismatches out of 20 contiguous nucleotides.
cDNAs are molecules that may be reverse-transcribed from fragments of message RNA from a genomic source. These fragments form a cDNA library of nucleic acid molecules. cDNA libraries are constructed from natural sources such as mammalian blood, semen, or tissue.
The term xe2x80x9csubtractive hybridizationxe2x80x9d refers to a method similar to cDNA cloning except that cDNA prepared from mRNA in unstimulated cells is added to mRNA in stimulated or different types of cells. cDNA/mRNA can then be precipitated to enrich the mRNA specific to the stimulation signal or different cell type.
The term xe2x80x9cPTP20 polypeptidexe2x80x9d refers to a polypeptide having an amino acid sequence preferably of at least 400 contiguous amino acids, more preferably of at least 450 contiguous amino acids, or most preferably of at least 453 contiguous amino acids set forth in SEQ ID NO: 7, or is substantially similar to such a sequence, or have substantially similar functional activities such as phosphatase activity or growth or differentiation activity, and may be measured as described herein, e.g. at p. 26, lines 9-25, or p. 29, lines 14-28. A sequence that is substantially similar will preferably have at least 90% identity (more preferably at least 95% and most preferably 99-100% identity) to the amino acid sequence of SEQ ID NO: 7. PTP20 polypeptides preferably have tyrosine phosphatase activity and fragments of the full length PTP20 sequence having such activity may be identified using techniques well known in the art, such as sequence comparisons and assays such as those described in the examples herein.
The term xe2x80x9cidentityxe2x80x9d refers to a property of sequences that measures their similarity or relationship. Identity is measured by dividing the number of identical residues by the total number of residues and multiplying the product by 100. Thus, two copies of exactly the same sequence have 100% identity, but sequences that are less highly conserved and have deletions, additions, or replacements may have a lower degree of identity. Those skilled in the art will recognize that several computer programs are available for determining sequence identity. Such programs are generally able to achieve maximum alignment by ignoring deletions or additions that would otherwise alter the calculation of the percentage of identity between two sequences.
A preferred embodiment concerns nucleic acid molecules relating to PTP20 enriched, isolated, or purified from a mammalian source. These nucleic acid molecules can be isolated from, among other sources, blood, semen, or tissue.
The term xe2x80x9cmammalianxe2x80x9d refers to such organisms as, for example, mice, fats, rabbits, goats, monkeys, apes, and preferably humans. Although the PTP20 nucleic acid molecule of SEQ ID NO:1 is isolated from rat cells, current recombinant DNA techniques can readily elucidate a related nucleic acid molecule in human tissue.
Another preferred embodiment concerns an isolated nucleic acid molecule relating to PTP20 that encodes at least twelve contiguous amino acids of the amino acid sequence set forth in SEQ ID NO: 7. Preferably at least 12, 15, 20, 25, 30, 35, 40, 50, 100, 200 or 300 contiguous amino acids of the PTP20 sequence are encoded. This preferred embodiment of the invention is achieved by applying routine recombinant DNA techniques known to those skilled in the art.
Another aspect of the invention features a nucleic acid probe that can detect nucleic acid molecules encoding a PTP20 polypeptide in a sample.
The term xe2x80x9cnucleic acid probexe2x80x9d refers to a nucleic acid molecule that is complementary to and can bind a nucleic acid sequence encoding the amino acid sequence substantially similar to that set forth in SEQ ID NO: 7.
By xe2x80x9csubstantially similarxe2x80x9d it is meant a sequence that will preferably have at least 90% identity (more preferably at least 95% and most preferably 99-100%) to the sequence set forth in SEQ ID NO: 1 or SEO ID NO: 7.
By xe2x80x9cidentityxe2x80x9d is meant a property of sequences that measures their similarity or relationship. Identity is measured by dividing the number of identical residues in the two sequences by the total number of residues and multiplying the product by 100. Thus, two copies of exactly the same sequence have 100% identity, but sequences that are less highly conserved and have deletions, additions, or replacements have a lower degree of identity. Those skilled in the art will recognize that several computer programs are available for determining sequence identity.
The nucleic acid probe or its complement encodes any one of the amino acid molecules set forth in the invention. Thus the nucleic acid probe can encode at least 12, 75, 90, 105, 120, 150, 200, 250, 300 or 350 contiguous amino acids of the full-length sequence set forth in SEQ ID NO: 7.
The nucleic acid probe can be labeled with a reporter molecule or molecules. The term xe2x80x9creporter moleculexe2x80x9d refers to a molecule that is conjugated to the nucleic acid probe or is contained within the nucleic acid probe. The reporter molecule allows the detection of the probe by methods used in the art. Reporter molecules are chosen from, but not limited to; the group consisting of an enzyme, such as a peroxidase, a radioactive element, or an avidin molecule.
A nucleic acid probe, whether labeled or unlabeled, should hybridize to a complement in a sample.
The nucleic acid probe of the present invention can be a nucleic acid molecule encoding a conserved or unique region of amino acids of PTP20. These nucleic acid molecules are useful as hybridization probes to identify and clone additional polypeptides relating to PTP20.
The term xe2x80x9cconserved nucleic acid regionsxe2x80x9d, refers to regions present in two or more nucleic acid molecules encoding a PTP20 polypeptide, to which a particular nucleic acid sequence can hybridize under low stringency conditions. Examples of low stringency conditions suitable for screening nucleic acid molecules encoding PTP20 polypeptides are provided in Abe, et al. J. Biol. Chem., 19:13361 (1992) (hereby incorporated by reference herein in its entirety, including any drawings). Preferably, conserved regions differ by no more than 5 out of 20 nucleotides. As mentioned above, protein tyrosine phosphatases share conserved regions in their extracellular and catalytic domains.
The term xe2x80x9cunique nucleic acid regionxe2x80x9d concerns a sequence present in a full length nucleic acid coding for a PTP20 polypeptide that is not present in a sequence coding for any other naturally occurring polypeptide. Such regions preferably comprise 30 or 45 contiguous nucleotides present in the full length nucleic acid sequence encoding a PTP20 polypeptide. In particular, a unique nucleic acid region is preferably of mammalian origin.
Methods for using the probes include detecting the presence or amount of PTP20 RNA in a sample by contacting the sample with a nucleic acid probe under conditions such that hybridization occurs. The nucleic acid duplex formed between the probe and a nucleic acid sequence coding for a PTP20 polypeptide may be used in the identification of the sequence of the nucleic acid detected (for example see, Nelson et al., in Nonisotopic DNA Probe Techniques, p. 275 Academic Press, San Diego (Kricka, ed., 1992) hereby incorporated by reference herein in its entirety, including any drawings). Kits for performing such methods may be constructed to include a container holding a nucleic acid probe.
In yet another aspect, the invention relates to a nucleic acid vector comprising a nucleic acid molecule encoding a PTP20 polypeptide and a promoter element effective to initiate transcription in a host cell.
The term xe2x80x9cnucleic acid vectorxe2x80x9d relates to a single or double stranded circular nucleic acid molecule that can be transfected or transformed into cells and replicate independently or within the host cell genome. A circular double stranded nucleic acid molecule can be cut and thereby linearized upon treatment with restriction enzymes. An assortment of vectors, restriction enzymes, and the knowledge of the nucleotide sequences that the restriction enzymes operate upon are readily available to those skilled in the art. A nucleic acid molecule of the invention can be inserted into a vector by cutting the vector with restriction enzymes and ligating the two pieces together.
Many techniques are available to those skilled in the art to facilitate transformation or transfection of the expression construct into a prokaryotic or eukaryotic organism. The terms xe2x80x9ctransformationxe2x80x9d and xe2x80x9ctransfectionxe2x80x9d refer to methods of inserting an expression construct into a cellular organism. These methods involve a variety of techniques, such as treating the cells with high concentrations of salt, an electric field, or detergent, to render the host cell outer membrane or wall permeable to nucleic acid molecules of interest.
The term xe2x80x9cpromoter elementxe2x80x9d describes a nucleotide sequence that is incorporated into a vector that, once inside an appropriate cell, can facilitate transcription factor and/or polymerase binding and subsequent transcription of portions of the vector DNA into mRNA. The promoter element precedes the 5xe2x80x2 end of the PTP20 nucleic acid molecule such that the latter is transcribed into mRNA. Host cell machinery then translates mRNA into a polypeptide.
Those skilled in the art would recognize that a nucleic acid vector can contain many other nucleic acid elements besides the promoter element and the PTP20 nucleic acid molecule. These other nucleic acid elements include, but are not limited to, origins of replication, ribosomal binding sites, nucleic acid sequences encoding drug resistance enzymes or amino acid metabolic enzymes, and nucleic acid sequences encoding secretion signals, periplasm or peroxisome localization signals, or signals useful for polypeptide purification.
A nucleic acid vector can be useful for identifying natural binding partners of PTP20 polypeptides.
The term xe2x80x9cnatural binding partnersxe2x80x9d refers to polypeptides that bind to PTP20 and play a role in propagating a signal in a signal transduction process. The term xe2x80x9cbinding partnerxe2x80x9d also refers to a polypeptide that binds to PTP20 within a cellular environment with high affinity. High affinity represents an equilibrium binding constant on the order of 10xe2x88x926 M. However, a natural binding partner can also transiently interact with a PTP20 polypeptide and chemically modify it. PTP20 natural binding partners are chosen from a group consisting of, but not limited to, src homology 2 (SH2) (Sadowski, et al, Mol. Cell. Biol. 6:4396, 1986; Pawson and Schlessinger, Curr. Biol. 3:434, 1993) or 3 (SH3) domains (Mayer, et al, Nature 332:272, 1988; Pawson and Schlessinger, Curr. Biol. 3:434, 1993), other phosphoryl tyrosine binding domains, and receptor and non-receptor protein kinases or protein phosphatases.
Methods are readily available in the art for identifying natural binding partners of polypeptides of interest by screening cDNA libraries included in one nucleic acid vector with a nucleic acid molecule encoding the desired polypeptide in another expression construct. Vojtek et al., 1993, Cell 74:205-214. These techniques often utilize two halves of a transcription factor, one of which is fused to a polypeptide encoded by the cDNA library, and the other of which is fused to the polypeptide of interest. Interactions between a polypeptide encoded by the cDNA library and the polypeptide of interest are detected when their interaction concomitantly brings together the two halves of the transcription factor and activates a gene that reports the interaction. Any of the nucleic molecules encoding PTP20 polypeptide can be readily incorporated into a nucleic acid vector used in such a screening procedure by utilizing standard recombinant DNA techniques in the art.
Another aspect of the invention relates to a recombinant cell or tissue comprising a nucleic acid molecule encoding a PTP20 polypeptide.
The term xe2x80x9crecombinantxe2x80x9d refers to an organism that has a new combination of genes or nucleic acid molecules. A new combination of genes or nucleic acid molecules can be introduced to an organism using a wide array of nucleic acid manipulation techniques available to those skilled in the art. The recombinant cell can be a eukaryotic or a prokaryotic organism.
The term xe2x80x9ceukaryotexe2x80x9d refers to an organism comprised of cells that contain a nucleus. Eukaryotes are differentiated from xe2x80x9cprokaryotesxe2x80x9d which do not house their genomic DNA inside a nucleus. Prokaryotes include unicellular organisms such as bacteria while eukaryotes are represented by yeast, invertebrates, and vertebrates.
The term xe2x80x9corganismxe2x80x9d relates to any living being comprised of at least one cell. An organism can be as simple as one eukaryotic cell or as complex as a mammal.
The recombinant cell can harbor a nucleic acid vector that is extragenomic. The term xe2x80x9cextragenomicxe2x80x9d refers to a nucleic acid vector which does not insert into the cell genome. Many nucleic acid vectors are designed with their own origins of replication allowing them to utilize the recombinant cell replication machinery to copy and propagate the vector nucleic acid sequence. These vectors are small enough that they are not likely to harbor nucleic acid sequences homologous to genomic sequences of the recombinant cell. Thus these vectors replicate independently of the host genome and do not recombine with or integrate into the genome.
A recombinant cell can harbor a portion of a nucleic acid vector in an intragenomic fashion. The term xe2x80x9cintragenomicxe2x80x9d defines a nucleic acid construct that is incorporated within the cell genome. Multiple nucleic acid vectors available to those skilled in the art contain nucleic acid sequences that are homologous to nucleic acid sequences in a particular organism""s genomic DNA. These homologous sequences will result in recombination events that integrate portions of the vector into the genomic DNA. Those skilled in the art can control which nucleic acid sequences of the vector are integrated into the cell genome by flanking the portion to be incorporated into the genome with homologous sequences in the vector.
Yet another aspect of the invention features an isolated, enriched, or purified PTP20 polypeptide.
The term xe2x80x9cisolatedxe2x80x9d, in reference to a polypeptide, describes a polymer of amino acids conjugated to one another, including polypeptides that are isolated from a natural source or that are synthesized. In certain aspects longer polypeptides are preferred, such as those with most of the contiguous amino acids set forth in SEQ ID NO: 7.
The isolated polypeptides of the present invention are unique in the sense that they are not found in a pure or separated state in nature. Use of the term xe2x80x9cisolatedxe2x80x9d indicates that a naturally occurring sequence has been removed from its normal cellular environment. Thus, the sequence may be in a cell-free solution or placed in a different cellular environment. The term does not imply that the sequence is the only amino acid chain present, but that it is essentially free (about 90-95% pure at least) of non-amino acid material naturally associated with it.
The term xe2x80x9cenrichedxe2x80x9d, in reference to a polypeptide, defines a specific amino acid sequence constituting a significantly higher fraction (2-5 fold) of the total of amino acids present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was separated. A person skilled in the art can preferentially reduce the amount of other amino acid sequences present, or preferentially increase the amount of specific amino acid sequences of interest, or both. However, the term xe2x80x9cenrichedxe2x80x9d does not imply that there are no other amino acid sequences present. Enriched simply means the relative amount of the sequence of interest has been significantly increased. The term xe2x80x9csignificantxe2x80x9d indicates that the level of increase is useful to the person making such an increase. The term also means an increase relative to other amino acids of at least 2 fold, or more preferably at least 5 to 10 fold, or even more. The term also does not imply that there are no amino acid sequences from other sources. Other source amino acid sequences may, for example, comprise amino acid sequences from a host organism: xe2x80x9cEnrichedxe2x80x9d is meant to cover only those situations in which a person has intervened to elevate the proportion of the desired amino acid sequence.
The term xe2x80x9cpurifiedxe2x80x9d, in reference to a polypeptide, does not require absolute purity (such as a homogeneous preparation); instead, it represents an indication that the amino acid sequence is relatively more pure than in a cellular environment. The concentration of the preferred amino acid sequence should be at least 2-5 fold greater (in terms of mg/ml) than its concentration in a cellular environment. Purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is preferred. The substance is preferably free of contamination, as indicated by purity levels of 90%, 95%, or 99%.
A preferred embodiment relates to a PTP20 polypeptide that is a unique fragment of a PTP20 polypeptide.
The term xe2x80x9cunique fragmentxe2x80x9d refers to a stretch of contiguous amino acids in PTP20 that is of a different sequence than another PTP. At least 12, 15, 20, 25, 30, 35, 40, 50, 100, 150, 200, 250, 300, or 350 contiguous amino acids of the full-length amino acid sequence of PTP20 are unique to PTP20.
The PTP20 polypeptide can be isolated, enriched, or purified from a prokaryotic or eukaryotic recombinant cell. A eukaryotic cell can arise from organisms including mammals and preferably humans. Multiple standard techniques are available to those skilled in the art to facilitate isolation, enrichment, or purification of a polypeptide from recombinant cells. These methods typically include lysing the recombinant cells and separating the polypeptide of interest from the rest of the cell polypeptides, nucleic acids, and fatty acid-based material using standard techniques known in the art.
Another aspect of the invention features an antibody that is monoclonal or polyclonal, or an antibody fragment having specific binding affinity to a PTP20 polypeptide.
Antibodies or antibody fragments are polypeptides which contain regions that can bind other polypeptides. The term xe2x80x9cspecific binding affinityxe2x80x9d describes an antibody that binds to a PTP20 polypeptide with greater affinity than it binds to other polypeptides under specified conditions.
The term xe2x80x9cpolyclonalxe2x80x9d refers to antibodies that are heterogenous populations of antibody molecules derived from the sera of animals immunized with an antigen or an antigenic functional derivative thereof. For the production of polyclonal antibodiwes, various host animals may be immunized by injection with the antigen. Various adjuvants may be used to increase the immunological reponse, depending on the host species.
xe2x80x9cMonoclonal antibodiesxe2x80x9d are substantially homogenous populations of antibodies to a particular antigen. They may be obtained by any techniques which provides for the production of antibody molecules by continuous cell lines in culture. Monoclonal antibodies may be obtained by methods known to those skilled in the art. See, for example, Kohler, et al., Nature 256:495-497 (1975), and U.S. Pat. No. 4,376,110.
The term xe2x80x9cantibody fragmentxe2x80x9d refers to a portion of an antibody, often the hypervariable region and portions of the surrounding heavy and light chains, that displays specific binding affinity for a particular molecule. A hypervariable region is a portion of an antibody that physically binds to the polypeptide target.
Antibodies or antibody fragments having specific binding affinity to a PTP20 polypeptide may be used in methods for detecting the presence and/or amount of a PTP20 polypeptide in a sample by probing the sample with the antibody under conditions suitable for PTP20-antibody immunocomplex formation and detecting the presence and/or amount of the antibody conjugated to the PTP20 polypeptide. Diagnostic kits for performing such methods may be constructed to include antibodies or antibody fragments specific for PTP20 as well as a conjugate of a binding partner of the antibodies or the antibodies themselves.
An antibody or antibody fragment with specific binding affinity to a PTP20 polypeptide can be isolated, enriched, or purified from a prokaryotic or eukaryotic organism. Routine methods known to those skilled in the art enable production of antibodies or antibody fragments, in both prokaryotic and eukaryotic organisms. Purification, enrichment, and isolation of antibodies, which are polypeptide molecules, are described above.
Another aspect of the invention features a hybridoma which produces an antibody having specific binding affinity to a PTP20 polypeptide. A xe2x80x9chybridomaxe2x80x9d is an immortalized cell line which is capable of secreting an antibody, for example an antibody with specific binding affinity to PTP20.
Another aspect of the invention features an isolated, enriched, or purified nucleic acid molecule comprising a nucleotide sequence that: (a) encodes a polypeptide having the full length amino acid sequence set forth SEQ ID NO: 7; (b) is the complement of the nucleotide sequence of (a); (c) hybridizes under highly stringent conditions to the nucleotide molecule of (b) and encodes a naturally occurring PTP20 protein; (d) encodes a PTP20 polypeptide having the full length amino acid sequence of the sequence set forth in SEQ ID NO: 7 except that it lacks one or more of the following segments of amino acid residues 1-58, 59-294, or 295-453. (e) is the complement of the nucleotide sequence of (d); (f) encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 7 from amino acid residues 1-58, 59-294, or 295-453; (g) is the complement of the nucleotide sequence of (f); or (h) encodes a polypeptide having the full length amino acid sequence set forth in SEQ ID NO: 7 except that it lacks one or more of the domains selected from the group consisting of a N-terminal domain, a catalytic domain, and a C-terminal domain, or (i) is the complement of the nucleotide sequence of (h).
The term xe2x80x9cN-terminal domainxe2x80x9d refers to a portion of the full length PTP20 amino acid sequence spanning from the amino terminus to the start of the catalytic domain. The N-terminal domain spans amino acid residues 1-58 of the sequence set forth in SEQ ID NO: 7.
The term xe2x80x9ccatalytic domainxe2x80x9d refers to a portion of the PTP20 amino acid molecule that does not contain the N-terminal domain and has catalytic activity. The catalytic domain spans amino acid residues 59-294 of the sequence set forth in SEQ ID NO: 7.
The term xe2x80x9cC-terminal domainxe2x80x9d refers to a portion of PTP20 that begins at the end of the catalytic domain and ends at thecarboxy terminal amino acid, which is the last amino acid encoded before the stop codon in the nucleic acid sequence. The C-terminal domain spans. amino acid residues 295-453 of the sequence set forth in SEQ ID NO: 7.
Domains are regions of polypeptides which have particular functions. For instance, N-terminal or C-terminal domains of signal transduction proteins can serve functions including, but not limited to, binding molecules that localize the signal transduction molecule to different regions of the cell or binding other signaling molecules directly responsible for propagating a particular cellular signal. Some domains can be expressed separately from the rest of the protein and function by themselves, while others must remain part of the intact protein to retain function. The latter are termed functional regions of proteins and also relate to domains.
Functional regions of PTP20 may be identified by aligning the amino acid sequence of PTP20 with amino acid sequences of other polypeptides with known functional regions. If regions of PTP20 share high amino acid identity with the amino acid sequences of known functional regions, then PTP20 can be determined to contain these functional regions by those skilled in the art. The functional regions can be determined, for example, by using computer programs and sequence information available to those skilled in the art.
Other functional regions of signal transduction molecules that may exist in the PTP20 amino acid sequence include, but are not limited to, proline-rich regions or phosphoryl tyrosine regions. These regions can interact with natural binding partners such as SH2 or SH3 domains of other signal transduction molecules.
In yet another aspect, the invention includes a nucleic acid vector containing a nucleic acid molecule described above.
Another aspect of the invention relates to a recombinant cell or tissue that contains a nucleic acid molecule described above.
In yet another aspect, the invention features a method of identifying compounds capable of modulating PTP20 catalytic activity. This method consists of the following steps: (a) adding a compound to cells containing a PTP20 polypeptide; and (b) detecting a change in the catalytic activity of the PTP20 polypeptide.
The term xe2x80x9cmodulatingxe2x80x9d refers to the ability of a compound to alter PTP20 catalytic activity. A modulator preferably activates PTP20 catalytic activity, more preferably activates or inhibits PTP20 catalytic activity depending on the concentration of the compound exposed to PTP20, or most preferably inhibits PTP20 catalytic activity.
The term xe2x80x9ccompoundxe2x80x9d includes small organic molecules including, but not limited to, oxindolinones, quinazolines, tyrphostins, quinoxalines, and extracts from natural sources.
The term xe2x80x9ca change in catalytic activityxe2x80x9d, in the context of the invention, defines a method of observing a change in PTP20 catalytic activity in response to adding a compound to cells. The catalytic activity of a PTP20 polypeptide can be detected, for example, by measuring the amount of a substrate, such as p-nitrophenylphosphate, converted to a product, such as p-nitrophenol, with respect to time. Addition of a compound to cells expressing a PTP20 polypeptide may either enhance (activate) or lower (inhibit) the catalytic activity. If a compound lowers PTP20 catalytic activity, the compound is assumed to bind to a PTP20 polypeptide and block the ability of PTP20 to bind and/or turn over a substrate. If a compound enhances PTP20 catalytic activity, the compound is assumed to bind to a PTP20 polypeptide and facilitate the ability of PTP20 to bind and/or turn over a substrate.
The method can utilize any of the molecules disclosed in the invention. These molecules include nucleic acid molecules encoding PTP20 polypeptides, nucleic acid vectors, recombinant cells, polypeptides, or antibodies of the invention.
Another aspect of the invention relates to a method of identifying compounds useful for diagnosis or treatment of an abnormal condition in an organism. The abnormal condition can be associated with an aberration in a signal transduction pathway characterized by an interaction between a PTP20 polypeptide and a natural binding partner. The method comprises the following steps: (a) adding a compound to cells; and (b) detecting whether the compound promotes or disrupts an interaction between a PTP20 polypeptide and a natural binding partner.
The term xe2x80x9cabnormal conditionxe2x80x9d refers to a function in an organism""s cells or tissue that deviate from a normal function in the cells or tissue of that organism. In the context of this aspect of the invention, abnormal conditions can be associated with, for example, cell proliferation. Cell proliferative disorders include, but are not limited to, cancers such as fibrotic and mesangial disorders, abnormal angiogenesis and vasculogenesis, slow wound healing rates, psoriasis, diabetes mellitus, and inflammation. Abnormal conditions can also be associated with cell differentiation. Cell differentiation disorders include, but are not limited to, neurodegenerative disorders, slow wound healing rates, and grafting tissue grafting techniques.
The abnormal condition can be diagnosed when the organism""s cells exist within the organism or outside of the organism. Cells existing outside the organism can be maintained or grown in cell culture dishes. For cells harbored within the organism, many techniques exist in the art to administer compounds, including (but not limited to) oral, parenteral, dermal, and injection applications. For cells outside of the patient, multiple techniques exist in the art to administer the compounds, including (but not limited to) cell microinjection techniques, transformation techniques, and carrier techniques.
The term xe2x80x9csignal transduction pathwayxe2x80x9d refers to the molecules that propagate an extracellular signal through the cell membrane to become an intracellular signal. This signal can then stimulate a cellular response. The polypeptide molecules involved in signal transduction processes are typically receptor and non-receptor protein kinases, receptor and non-receptor protein phosphatases, and transcription factors.
The term xe2x80x9caberrationxe2x80x9d, in conjunction with a signal transduction process, refers to a polypeptide, for example PTP20, that is over- or under-expressed in an organism, mutated such that its catalytic activity is lower or higher than wild-type polypeptide, mutated such that it can no longer interact with a binding partner, is no longer modified by another protein kinase or protein phosphatase, or no longer interacts with a binding partner.
The term xe2x80x9cinteractionxe2x80x9d defines the complex formed between a PTP20 polypeptide and a natural binding partner. Compounds can bind to either the PTP20 polypeptide or the natural binding partner and disrupt the interaction between the two molecules.
The term xe2x80x9cpromote or disrupt the abnormal interactionxe2x80x9d refers to a method that can be accomplished by administering a compound to cells or tissues in an organism. A compound can promote an interaction between PTP20 and natural binding partners by forming favorable interactions with multiple atoms at the complex interface. Alternatively, a compound can inhibit an interaction between a protein kinase and natural binding partners by compromising favorable interactions formed between atoms at the complex interface.
Methods of detecting the ability of a compound to disrupt or enhance an interaction between PTP20 and a natural binding partner exist in the art. These methods include, but are not limited to, determining the effect of the compound upon the catalytic activity of a PTP20 polypeptide, the phosphorylation state of the PTP20 polypeptide or a natural binding partner, the ability of PTP20 to bind a natural binding partner, or a difference in a cell morphology. Differences in cell morphology include growth rates and differentiation rates of cells. These phenomena are simply measured by methods in the art. These methods typically involve observing the number of cells or the appearance of cells under a microscope with respect to time (for example, days).
The method can be performed in vitro as well as in vivo. In vivo applications include introducing a group of cells to an organism and then determining the effect of a compound administered to the organism on the state of the organism as well as the introduced cells. The art contains multiple methods of introducing a group of cells to an organism as well as methods of administering a compounds to an organism. The organism is preferably an animal such as a frog, mouse, rat, rabbit, monkey, or ape, and also a human.
Another aspect of the invention relates to a method of diagnosing an abnormal condition associated with cell proliferation or cell differentiation in an organism. The abnormal condition can be associated with an aberration in a signal transduction pathway characterized by an interaction between a PTP20 polypeptide and a natural binding partner. The method comprises the step of detecting an abnormal interaction.
The term xe2x80x9cdetecting an abnormal interactionxe2x80x9d defines a method of identifying a PTP20 molecule with an aberration in its activity. Detection is accomplished by using an antibody or antibody fragment of the invention, a nucleic acid probe of the invention, or a compound of the invention.
Techniques used in the art that incorporate this method include in vitro, in vivo, and in situ hybridization techniques. These techniques utilize nucleic acid probes of the invention.
A preferred embodiment of the invention is the diagnosis method relating to an organism that is a mammal.
The summary of the invention described above is not limiting and other features and advantages of the invention will be apparent from the following detailed description of the invention, and from the claims.